The MHC locus contributes significantly to the genetic risk for type 1diabetes (T1D), but the molecular mechanisms are not well understood. The central problem is that current experimental methods for characterization of self-reactive T cell populations are highly inadequate. These approaches, including tetramer labeling, intracellular cytokine staining (ICS) and ELISpot assays, enable sensitive detection of high- affinity microbe-specific T cell populations but are suboptimal for self-reactive T cells which tend to have low affinities for their peptide-MHC ligands. We will use a novel single-cell technology that enables sensitive detection of self-reactive T cells and generates a comprehensive body of data on surface phenotype, cytokine release and other functions, such as proliferation and cytotoxicity. A dense, elastomeric array of wells with subnanoliter volumes (nanowells) is generated by replica molding, and individual T cells are co-cultured with autologous mature dendritic cells pulsed with antigen. Many different cytokines are captured on a glass slide and quantified on a microarray scanner, while CD8 T cell cytotoxicity is quantified by imaging lysis of co- cultured target cells. T cells of interest can also be isolated for subsequent clonal expansion. This system provides a rapid and high-throughput method for ex vivo characterization of lymphocytes. Preliminary data show that this approach greatly increases the sensitivity of detection for self-reactive T cells and enables comprehensive assessment of their ex vivo functions. We will use this novel technology to address three fundamental questions on the mechanisms by which MHC genes confer susceptibility and resistance to T1D. First, it remains unknown whether there are important differences in CD4 and/or CD8 T cell functions in patients with distinct MHC haplotypes that confer different degrees of risk. We will compare cytokine patterns and CD8 T cell cytotoxicity in response to B cell antigens in patients who carry either DR3-DQ2 (DQ2) or DR4- DQ8 (DQ8) haplotypes or the highest risk DQ2/DQ8 haplotype. Distinct, yet complementary functions could account for the high risk conferred by heterozygosity for DQ2 and DQ8 haplotypes. Second, genetic data suggest that DQ trans-dimers (encoded in trans by different haplotypes) contribute to the high risk of patients with heterozygosity for DQ2/DQ8 haplotypes. We will directly test this hypothesis by cloning CD4 T cells from nanowells and testing their MHC-peptide specificity. Third, several haplotypes are known to confer dominant protection from T1D. Particularly important is the DR15-DQ6 haplotype because it reduces risk more than 30- fold and is common in populations with a high incidence of T1D. We will assess three possible mechanisms for dominant protection: epitope capture, deletion of particular effector T cell populations, or induction of b cell- specific T cels with regulatory functions that can control effector T cell responses. This highly novel approach will thus allow us to address central questions on the function of MHC genes in T1D. PUBLIC HEALTH RELEVANCE: This project focuses on the mechanisms for MHC-linked susceptibility to T1D. A novel single cell approach will be used to interrogate the function of cell specific CD4 and CD8 T cell populations in patients and normal subjects with different predisposing or protective MHC genotypes. These studies have the potential to impact the design of future prevention approaches and to improve prediction of T1D in susceptible populations.